The structures of bacteriophage P22 repressor and bacteriophage 434 repressor and Cro are known to high resolution. Moreover, the structures of 434 repressor and Cro in complex with at least three different operators have been determined. This set of proteins therefore, forms an excellent model system in which biochemical and structural data can be compared. The binding of the 434 and P22 proteins affect, and are affected by, the conformation and deformability of noncontacted bases at the centers of their binding sites. This effect and the wealth of structural and biochemical information makes these proteins ideal candidates for exploring, in detail, both the direct and indirect effects of DNA sequence on recognition of specific binding sites by proteins. The role of sequence dependent differences in DNA twist, twisting flexibility, and major or minor groove geometry in modulating the stability of complexes between 434 repressor and Cro and P22 repressor and their respective binding sites will be examined by a combination of biochemical and in vitro genetic methods. The idea that 434 repressor's operator position 4 base preferences are modulated by central sequence-dependent differences in the global configuration of the repressor-operator complex will be tested. The base sequence and structural foundations underlying the central sequence context effects on the base specific contacts between 434 repressor and operator position 4 will be determined. Comparing the sensitivity of Ala33-434 repressor and/or 434 Cro to position 4 substitution in OR1 and OR3 sequence contexts will allow examination of the role of specific contacts to position 4 in the central sequence context effect. By introducing mutations into the dimer interface of 434 repressor, the role of dimer interface flexibility in determining the sensitivity of 434 repressor to the central base composition can be examined. These mutant proteins will also be used to assess the role of the dimer interface in directing central sequence effects on repressor recognition of the position 4 base. By defining the optimal binding site for wild-type P22 repressor and comparing the sequence of this binding site to that preferred by P22 repressor molecules bearing mutations in their DNA contacting alpha- helices will allow determination of the pattern of amino acid-base pair contacts in P22 repressor-operator complexes. The relative influence of central sequence-directed differences in groove width and operator twist on noncontacted base-dependent alterations in affinity of P22 operator for P22 repressor will be examined. By investigating the effect of central bases changes on the ability of P22 repressor to specifically contact bases in the operator will enable us to determine whether the noncontacted base exert their effect by altering the strength of specific protein-DNA contacts.